There is a concern that there may be unregistered stocks of smallpox that can be used for bioterrorism or biological warfare. According to the WHO Advisory Committee on Variola Research, there is a need to develop strategies to treat smallpox infections should they reappear. It would also be important to have an effective drug at hand for the treatment of monkeypox disease in humans. We show here that 5-iodo-2'-deoxyuridine (IDU) is a potent inhibitor of vaccinia virus (VV) replication and that IDU inhibits VV DNA synthesis in a dose-dependent way. The in vivo protective effect of IDU was assessed in the VV tail lesion model in immunocompetent mice and in a lethal model for VV infection in SCID (severe combined immune deficiency) mice that had been infected either intranasally, intraperitoneally, or intravenously. Subcutaneous treatment with IDU at 150 and 100 mg/kg of body weight markedly reduced the number of tail lesions in immunocompetent NMRI mice. Untreated intranasally VV-infected SCID mice died at 20.8 +/- 3.1 days after infection (mean +/- standard deviation). Treatment with IDU (subcutaneously, 150 mg/kg/day [from day 0 to 4] and 75 mg/kg/day [from day 6 to 11]) delayed-virus induced mortality by 15 days (mean day of death +/- standard deviation, 35.8 +/- 6.7; P < 0.0001). This protective effect was associated with (i) an improvement of lung histology and (ii) a marked reduction in lung viral titers. IDU also delayed VV-induced mortality when mice had either been infected intraperitoneally or intravenously. Even when the start of treatment with IDU (in intraperitoneally VV-infected mice) was postponed until 2 or 4 days after infection, an important delay in virus-induced mortality was noted.